1. Field of the Invention
The present invention relates to an on-line base sequencing apparatus which comprises a gel electrophoresis apparatus for electrophoresing nucleic acid fragments such as DNA fragments treated by the Sanger method through electrophoresis lanes provided for the respective types of end bases thereby determining the base sequences of nucleic acids from the order of elution.
2. Description of the Background Art
An on-line base sequencing apparatus detects labeled DNA fragments, which are treated by the Sanger method, in order of elution, for detecting the DNA fragments eluted from electrophoresis gels. While study has been performed by using labeling materials of radioisotopes, which have been used in an off-line method, the apparatus is increased in size with inconvenience in handling in this case. Thus, generally carried out are a method of using fluorescent labels (refer to Nature, 1986, Vol. 321, pp. 674-679) and a method of using stable isotope labels (refer to Japanese Patent Laying-Open Gazette No. 2-176552 (1990)).
In such a base sequencing method using no radio isotopes, it is necessary to partially label DNAs with fluorescent materials or isotopes in advance of pretreatment by the Sanger method. Depending on the labeling method (or materials or portions), however, the Sanger reaction itself is influenced, and it is impossible to use specific primers and other chemical materials which have been used in the method employing radioisotopes.
There has recently been reported an attempt for electrophoresing DNA fragments labeled with neither fluorescent materials nor isotopes through gel filled capillaries and irradiating the DNA fragments with ultraviolet rays during such electrophoresis to excite the same, for detecting the as-generated fluorescence (refer to Anal. Chem., Vol. 65, No. 2, pp. 153-157 (1993)). In this case, however, the background is increased due to fluorescence also generated from the gels, while it is difficult to detect the fluorescence generated from the DNA fragments since the gels absorb such fluorescence. Although the capillaries are strongly acidified or alkalified in order to increase fluorescence efficiency of the DNA fragments, the DNA fragments are disadvantageously decomposed when the same are electrophoresed through strongly acidic gels, for example, and it is impossible to maintain the states of the DNA fragments which have been prepared by the Sanger method for the respective end bases. In such electrophoresis using strongly acidified gel filled capillaries, therefore, it is impossible to determine base sequences of DNAs even if fluorescence from unlabeled DNA fragments can be detected in high sensitivity.